PubMed · 7532269
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G F Babcock, S M Dawes. 1994. Immunophenotyping using fixed cells.. https://doi.org/10.1016/s0091-679x(08)61710-0
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Ruthenium tetroxide and osmium tetroxide were compared as post-fixatives in the preparation of human epidermis for transmission electron microscopic examination. Both reagents revealed characteristic lamellar granules within the granular layer and extruded lamellar granule contents in the upper granular layer. The transformation of the granule contents into multilamellar sheets at the interface between the granular and cornified layers and the persistence of these sheets through all levels of the stratum corneum were demonstrated only with ruthenium tetroxide fixation. Therefore, the reactivity of osmium tetroxide with isolated epidermal lipids was examined. The failure of osmium tetroxide to reveal membrane structures in the stratum corneum can be explained by its inability to react with many of the lipid components of these membranes, rather than to selective removal of lipids during tissue processing, as was formerly believed. Ruthenium tetroxide, a stronger oxidizing agent than osmium tetroxide, overcomes this problem but has other severe limitations as a post-fixative.
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Whole blood lysis and fixation methods for flow cytometric (FCM) analysis were tested for their ability to reduce the infectivity of human T-lymphotropic virus type 1 (HTLV-I). Our goals were to: (1) determine the effects of 1.0 and 2.0% paraformaldehyde (PF) fixation on HTLV-I infected cell lines and (2) assess the infectivity of blood samples containing HTLV-I-infected cells following processing with 5 commercially available products (Immuno-lyse, ImmunoPrep/Q-Prep, FACS lysis solution, GenTrak lyse and fix reagent, and Ortho-mune lysing reagent) compared to ammonium chloride lysis with either 0.1 or 1.0% PF fixation. Infectivity was determined by monitoring HTLV-I p24 antigen production in cocultures of treated leukocytes with uninfected peripheral blood mononuclear cells (PBMC). Each method effectively reduced the viability of treated leukocytes. Commercial lysis/fixation methods significantly reduced HTLV-I infectivity compared to prepared ammonium chloride/PF-based methods. For all preparations, increasing the time of fixation (e.g., 60 min) effectively reduced viral infectivity. Taken together, these data suggest that commercially available fixatives greatly reduce, but do not eliminate the risk of HTLV-I infection during processing of viral-infected cells for FCM analysis.